Blubber and skin are commonly used tissues in stable isotope analysis for the purpose of investigating cetacean diet. Critical comparison of tissue-specific isotopic signals is, however, lacking resulting in uncertainty surrounding the representativeness and therefore utility of different tissues for accurate determination of recent foraging. Paired blubber and skin biopsy samples were collected from free-swimming southern hemisphere humpback whales in SE Queensland Australia between 2008 and 2018 as part of long-term monitoring under the Humpback Whale Sentinel Program. A total of 171 paired biopsies were included in this study, were the authors recorded migration direction (north or south) and sex (male or female). Both tissues were treated for lipid correction: blubber was applied a solvent lipid extraction; a modified methanol-dichloromethane-water method, whilst skin was applied a mathematical mass-balance model for lipid correction. Significant differences were observed for both δ13C and δ15N between lipid-extracted blubber and lipid-corrected skin tissue, flagging previously undocumented methodological considerations, and the need for method validation and standardisation in application of these approaches. This study therefore advances methodological aspects of cetacean dietary analysis. This is of elevated importance in the context of rapidly changing ocean ecosystems.